The present invention relates to the imaging of multiwell plates, in particular, to the use of a doubly telecentric lens and system for accurate and efficient detection of low levels of light from high density multiwell plates.
Microtiter or multi-well plates are becoming increasingly popular in various chemical and biological assays. Further, high-density format plates, such as 384, 864 and 1536 well plates, are beginning to displace 96-well plates as the plate of choice. Many of the assays conducted in multiwell plates employ some type of light detection from the plate as the reporter for positive or negative assays results. Such assays include fluorescence assays, luminescence assays (e.g., luciferase-based assays), phosphorescence assays, scintillation assays, and the like. In particular, with the advent of solid phase scintillating materials, capsules and beads, homogeneous scintillation proximity assays (SPA)-are now being performed with increasing frequency in multiwell plates.
Detection of light signals from multiwell plates in the past has typically been done using plate readers, which generally employ a photodetector, an array of such photodetectors, photomultiplier tubes or a photodiode array to quantify the amount of light emitted from different wells. Such plate readers have been disclosed, for example, by Russell, et al., U.S. Pat. No. 4,810,096, issued Mar. 7, 1989, and VanCauter, et al., U.S. Pat. No. 5,198,670, issued Mar. 30, 1993. Although plate readers can detect the total light from each well, they have a number of limitations. For example, plate readers are typically not capable of resolving discrete light sources in a single well, so they could not be used, for example, to differentiate light from different beads in one well. Further, most plate readers have fewer photodetectors than there are wells in the plate, so at least some wells must be read serially, adding to the time required to complete the assays. This becomes a substantial problem in assays where the light signal is so low that each well needs to be in the detection field for an extended period of time (e.g., tens of minutes). In addition, most currently-available plate readers have been designed for 96-well plates. Although some can be adapted for, e.g., 384-well plates, the adaptation does not result in any significant increase in throughput, since a 384-well plate going through a modified 96-well reader typically takes four times as long to read as a 96-well plate.
Another technique that has been applied to the detection of light from multiwell plates is imaging. Prior art imaging systems typically comprise a standard 50-55 mm f1.4 photographic lens coupled to a camera. While such systems can be used to image an entire multiwell plate, and theoretically provide resolution of discrete light points within individual wells, they have poor sensitivity, even when coupled to efficient cameras, so that many assays still require imaging times of tens of minutes or more. Other assays, such as SPA bead-based assays, cannot be performed at all due to lack of sensitivity. Further, images acquired with such systems suffer from vignetting and lateral distortion effects, making it difficult or impossible to compare signals from center portions of the plate with signals from lateral wells.
The present invention provides lenses and systems which overcome the above-described disadvantages of prior art methods of light detection in multiwell plates. In particular, the present invention provides, for the first time, a doubly telecentric lens-based system with the ability to image SPA bead-based assays The telecentric lens of the invention is economical to manufacture due to a design employing a minimal total number of lens elements, the use of spherical lens elements, and generous tolerance limits in lens fabrication. Further, the telecentric lens of the present invention is the first such lens to provide an unprocessed image of a multiwell plate that is substantially free from vignetting, chromatic aberration and distortion.
In one aspect, the present invention includes a multiple element doubly telecentric lens for imaging a substantially flat object onto an image plane. The lens is preferably color-corrected, particularly in the range of 400 nm-700 nm, and comprises, in succession from a side of incident light, the following lens elements or groups (illustrated in FIGS. 2 and 3): (i) a biconvex field lens element L1, (ii) separated by a distance D from the field lens element, a positive meniscus lens element L2, concave toward the incident light side, (iii) a double-Gauss lens element group, (iv) a positive meniscus lens element L10, convex toward the incident light, (v) a positive meniscus lens element L11, convex toward the incident light, and (vi) a piano concave field flattener lens element L12, concave toward the incident light side. Changing distance D changes magnification of the telecentric lens.
In a preferred embodiment, the double-Gauss lens element group comprises, in succession from the side of incident light, (i) an incident-side lens element group, comprising, in succession from the side of incident light, (a) a biconvex lens element L3, (b) a positive meniscus lens element L4, convex toward the incident light, and (c) a doublet formed of two positive meniscus lens elements, L5 and L6, both convex toward the incident light; (ii) an aperture stop; and (iii) an image-side lens element group, comprising, in succession from the side of incident light, (a) a doublet formed of a biconcave lens element L7 and a biconvex lens element L8, the doublet being concave toward the incident light, and (b) a biconvex lens element.
The adjacent surfaces of the above-listed lens elements are preferably separated by distances as follows: between about 11.4xe2x80x3 and about 13.4xe2x80x3 between element L1 and element L2; about 3.9xe2x80x3 between element L2 and element L3; about 0.3xe2x80x3 between element L3 and element L4; about 0.04xe2x80x3 between element L4 and element L5; about 0.0xe2x80x3 between element L5 and element L6; about 2.3xe2x80x3 between element L6 and element L7; about 0.0xe2x80x3 between element L7 and element L8; about 0.1xe2x80x3 between element L8 and element L9; about 0.1xe2x80x3 between element L9 and element L10; about 0.1xe2x80x3 between element L10 and element L11; and about 0.3xe2x80x3 between element L11 and element L12.
Further, the lens described above preferably has the following characteristics, where elements L1-L12 are made of glass and have surfaces S1-S24; each of the surfaces is convex (CX), concave (CC) or Plano (XX); and the CX and CC surfaces have a radius measured in inches:
In another embodiment, the telecentric lens is designed to form a focused image at the image plane when the field lens element (L1) is positioned between about 20 mm and about 30 mm, preferably about 25 mm from the object. In other embodiments, distance D is preferably between about 11.4xe2x80x3 and about 13.4xe2x80x3; the lens has a numerical aperture of between about 0.5 and 0.6, preferably about 0.55; the lens has a magnification of between about xe2x88x920.20 and xe2x88x920.25, preferably about xe2x88x920.224; and the lens has a field of view of between about 4xe2x80x3 and about 6xe2x80x3, preferably about 5xe2x80x3 in diameter.
In another aspect, the invention includes a multiple element telecentric lens for imaging a multiwell plate having square wells onto a CCD pixel array. The lens has a magnification that results in each well of the plate mapping to an integer number of pixels in the CCD array. In one embodiment, the CCD array is a 1024xc3x971024 array. In a related embodiment, the multiwell plate has dimensions of a standard multiwell plate with a 2:3 aspect ratio of rows:columns, and all wells in a single row are imaged onto a rectangular region of the 1024xc3x971024 CCD array, the region being 1008 pixels in length. In another embodiment, the multiwell plate is selected from the group consisting of a 384-well plate, an 864-well plate, and a 1536-well plate.
Still another aspect of the invention includes a multiple element doubly telecentric lens for imaging a substantially flat object onto an image plane. The lens is preferably color corrected and has the following characteristics: (i) between 12 and 16 lens elements, preferably 12; (ii) a numerical aperture of between about 0.5 and 0.6, preferably 0.55; (iii) less than about 5% vignetting, preferably substantially zero vignetting; (iv) less than about 0.05% distortion; (v) a magnification of between about xe2x88x920.20 and about xe2x88x920.25, preferably between about xe2x88x920.22 and about xe2x88x920.23, more preferably about xe2x88x920.225; and (vi) a field of view of between about 4xe2x80x3 and about 6xe2x80x3, preferably about 5xe2x80x3 in diameter.
In one embodiment, the telecentric lens is used for imaging a multiwell plate having square wells onto a CCD pixel array, and has a magnification that results in each well of the plate mapping to an integer number of pixels in the CCD array.
Also included as part of the invention is a system for imaging a plate having a plurality of wells. The system comprises: (i) a multiple element telecentric lens suitable for imaging multiwell plates (e.g., as described above), (ii) a camera operably connected to the lens, and (iii) a robot having a base member and at least one arm, wherein the arm includes a grasping mechanism which is adapted to grasp the plate, and wherein the grasping mechanism is configured to receive the plate in a repeatable and known location such that the location of each well relative to the grasping mechanism is known by the robot.
In a related aspect, the invention includes a system for imaging a standard sample plate. The system includes a multiple element doubly telecentric lens as described above, a camera operably connected to the lens, and a chamber for receiving the plate during imaging. In one embodiment, the camera is a cooled 1024xc3x971024 CCD array camera. In another embodiment, the telecentric lens and the camera are mounted on a slide support such that magnification of the lens and focus of the camera can be adjusted separately.
In yet another embodiment, the system further comprises a means for positioning the plate in the chamber, such as a robot, conveyer belt, or the like. A system using a robot preferably further comprises a plurality of stations at known locations relative to the robot, and further includes a processor associated with the robot, where the processor is configured to control movement of the robot to place the plate at predetermined locations at the stations. The standard sample plate preferably has having a plurality of wells, the robot preferably has a base member and at least one arm, the arm preferably includes a grasping mechanism which is adapted to grasp the plate, and the grasping mechanism is preferably configured to receive the plate in a repeatable and known location such that the location of each well relative to the grasping mechanism is known by the robot.
In still another embodiment, the system further comprises a translation mechanism for axially translating the frame member, wherein the grasping mechanism comprises a frame member having edges which are adapted to frame at least a portion of a periphery of the plate in a repeatable and predictable manner so that the location of the wells of the plate relative to the frame member is known when the plate is received into the frame member; and a clamping arm that is pivotally attached to the frame member and which is adapted to engage a portion of the periphery of the plate to secure the plate to the frame member when the plate is received within the frame member.
The invention further includes a method for imaging a multiwell plate. The method comprises the steps of (i) positioning the plate under a multiple element doubly telecentric lens such as is described above, (ii) collecting light from the plate with the telecentric lens, (iii) transmitting the light through the lens to an image detection device, and (iv) using output from the image detection device to generate an image of the multiwell plate. In one embodiment, the image detection device is a CCD camera.
Also part of the invention is a method for imaging a solid-phase scintillant used in a scintillation proximity assay (SPA) in a standard multiwell plate. The method includes the steps of (i) positioning the plate under a multiple element telecentric lens suitable for imaging the multiwell plate, for example, a lens such as is described above, (ii) collecting light from the plate with the telecentric lens, (iii) transmitting the light through the lens to an image detection device, and (iv) using output from the image detection device to generate an image of the multiwell plate. In one embodiment, the image detection device is a (preferably cooled) CCD camera.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings.